PKCδ Inhibitor Research Articles

Hormesis of low-dose inhibition of pAkt1 (Ser473) followed by a great increase of proline-rich inositol polyphosphate 5-phosphatase (PIPP) level in oocytes.

Hormesis describes a biphasic dose-response relationship generally characterized by a low-dose excitement and a high-dose inhibition. This phenomenon has been observed in the regulation of cell, organ, and organismic level. However, hormesis has not reported in oocytes. In this study, we observed, for the first time, hormetic responses of PIPP levels in oocytes by inhibitor of Akt1 or PKCδ. The expression of PIPP was detected by qPCR, immunofluorescent (IF), and Western Blot (WB). To observe the changes of PIPP levels, we used the inhibitors against pAkt1 (Ser473) or PKCδ, SH-6 or sotrastaurin with low and/or high-dose, treated GV oocytes and cultured for 4h, respectively. The results showed that PIPP expression was significantly enhanced when oocytes were treated with SH-6 or sotrastaurin 10μM, but decreased with SH-6 or sotrastaurin 100μM. We also examined the changes of PIPP levels when GV oocytes were treated with exogenous PtdIns(3,4,5)P3 or LY294002 for 4h. Our results showed that PIPP level was enhanced much higher under the treatment of 0.1μM PtdIns(3,4,5)P3 than that of 1μM PtdIns(3,4,5)P3, which is consistent with the changes of PIPP when oocytes were treated with inhibitors of pAkt1 (Ser473) or PKCδ. In addition, with PIPP siRNA, we detected that down-regulated PIPP may affect distributions of Akt, Cdc25, and pCdc2 (Tyr15). Taken together, these results show that the relationships between PIPP and Akt may follow the principle of hormesis and play a key role during release of diplotene arrest in mouse oocytes.

Cypermethrin Induces the Activation of Rat Primary Microglia and Expression of Inflammatory Proteins.

Cypermethrin activates microglia, which is found to be decisive in neurodegeneration in the experimental rats. While the involvement of microglial activation in toxicant-induced neurodegeneration is reported, the effect of low concentration of cypermethrin on the expression of inflammatory proteins from the rat primary microglia is not yet properly understood. The study intended to delineate the effect of low concentration of cypermethrin on the expression and release of proteins from the microglia. Rat primary microglial cells were treated with cypermethrin to check the expression of inflammatory proteins. Cypermethrin-treated microglia conditioned media and cells were collected to measure the expression and release of inflammatory proteins. Cypermethrin augmented the protein kinase C-δ (PKC-δ), inducible nitric oxide synthase (iNOS), phosphorylated mitogen-activated protein kinase (MAPK) p38 and p42/44, matrix metalloproteinase (MMP)-3, and MMP-9 levels in the cell lysate and tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) levels in the microglia conditioned media. Pre-treatment with minocycline, a microglial activation inhibitor or rottlerin, a PKC-δ inhibitor, notably reduced the release of TNF-α in the conditioned media and expression of iNOS protein in the microglia. Minocycline reduced the expression of PKC-δ, phosphorylated p38 and p42/44 MAPKs, MMP-3, and MMP-9 proteins in the microglia. While cypermethrin-treated conditioned media induced the toxicity in the rat primary neurons, minocycline or rottlerin reduced the cypermethrin treated microglia conditioned media-induced toxicity. The outcomes of the present study suggest that cypermethrin activates microglia and releases TNF-α and IL-1β as well as up-regulates the expression of PKC-δ, iNOS, phosphorylated p38 and p42/44 MAPKs, MMP-3, and MMP-9 proteins, which could contribute to neurodegeneration.

Blockade of anti-dsDNA ameliorates systemic lupus erythematosus in MRL/Faslpr mice through ameliorating inflammation via the PKCδ-NLRC4 axis.

Anti-double-stranded DNA (anti-dsDNA) is closely associated with the inflammatory burden in the brain after ischemic stroke. Here, we studied the inflammatory cascade and investigated the mechanisms behind the pro-inflammatory role of dsDNA in systemic lupus erythematosus (SLE). The serum levels of interleukin-1beta (IL-1β) and IL-6 in SLE patients and the corresponding controls were evaluated using ELISA, and the expression level of caspase-1 was evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). We found that the serum levels of IL-1β and IL-6 were increased in the SLE patients. The expression of caspase-1 was upregulated and positively correlated with the levels of pro-inflammatory factors. The level of anti-dsDNA was also elevated and positively correlated with the results for the mean fluorescence intensity (MFI) of caspase-1. Additionally, we evaluated the functions of PRKCD encoding protein kinase c delta (PKCδ) and NLRC4, in vivo, in MRL/Faslpr mice. We found that renal injury was aggravated, and the levels of pro-inflammatory factors were increased in the MRL/Faslpr mice. We also found that increased levels of NLRC4 in the mice exacerbated renal injury and increased the levels of pro-inflammatory factors, whereas inhibition of PKCδ had the opposite results. These findings provide unique perspectives on pathogenesis of SLE and indicate that inhibition of anti-dsDNA could attenuate renal inflammatory burden, representing a promising therapeutic opportunity for SLE.

Fine tuning by protein kinases of CaV1.2 channel current in rat tail artery myocytes

Seventeen compounds, rather selective, direct or indirect inhibitors and activators of PKA, PKG, and PKC, were analysed for effects on vascular CaV1.2 channel current (ICa1.2) by using the patch-clamp technique in single rat tail artery myocytes. The aim was to investigate how PKs regulate ICa1.2 and disclose any unexpected modulation of CaV1.2 channel function by these agents. The cAMP analogues 8-Br-cAMP and 6-Bnz-cAMP partially reduced ICa1.2 in dialysed cells, while weakly increasing it under the perforated configuration. The β-adrenoceptor agonist isoproterenol and the adenylate cyclase activator forskolin concentration-dependently increased ICa1.2; this effect was reversed by PKA inhibitors H-89 and KT5720, but not by PKI 6-22. The cGMP analogue 8-Br-cGMP, similarly to the NO-donor SNP, moderately reduced ICa1.2, this effect being reversed to a slight stimulation under the perforated configuration. Among PKG inhibitors, Rp-8-Br-PET-cGMPS decreased current amplitude in a concentration-dependent manner while Rp-8-Br-cGMPS was ineffective. The non-specific phosphodiesterase inhibitor IBMX increased ICa1.2, while H-89, KT5720, and PKI 6-22 antagonized this effect. The PKC activator PMA, but not the diacylglycerol analogue OAG, stimulated ICa1.2 in a concentration-dependent manner; conversely, the PKCα inhibitor Gö6976 markedly reduced basal ICa1.2 and, similarly to the PKCδ (rottlerin) and PKCε translocation inhibitors antagonised PMA-induced current stimulation. The ensemble of findings indicates that the stimulation of cAMP/PKA, in spite of the paradoxical effect of both 8-Br-cAMP and 6-Bnz-cAMP, or PKC pathways enhanced, while that of cGMP/PKG weakly inhibited ICa1.2 in rat tail artery myocytes. Since Rp-8-Br-PET-cGMPS and Gö6976 appeared to block directly CaV1.2 channel, their docking to the channel protein was investigated. Both compounds appeared to bind the α1C subunit in a region involved in CaV1.2 channel inactivation, forming an interaction network comparable to that of CaV1.2 channel blockers. Therefore, caution should accompany the use of these agents as pharmacological tools to elucidate the mechanism of action of drugs on vascular preparations.

Sepsis and Cerebral Dysfunction: BBB Damage, Neuroinflammation, Oxidative Stress, Apoptosis and Autophagy as Key Mediators and the Potential Therapeutic Approaches.

Sepsis-associated cerebral dysfunction is complex pathophysiology, generated from primary infections that are developed elsewhere in the body. The neonates, elderly population and chronically ill and long-term hospitalized patients are predominantly vulnerable to sepsis and related cerebral damage. Generally, electrophysiological recordings, severity and sedation scales, computerized imaging and spectroscopy techniques are used for its detection and diagnosis. About the underlying mechanisms, enhanced blood-brain barrier permeability and metalloprotease activity, tight junction protein loss and endothelial cell degeneration promote the influx of inflammatory and toxic mediators into the brain, triggering cerebrovascular damage. An altered neutrophil count and phenotype further dysregulate the normal neuroimmune responses and neuroendocrine stability via modulated activation of protein kinase C-delta, nuclear factor kappa-B and sphingolipid signaling. Glial activation, together with pro-inflammatory cytokines and chemokines and the Toll-like receptor, destabilize the immune system. Moreover, superoxides and hydroperoxides generate oxidative stress and perturb mitochondrial dynamics and ATP synthesis, propagating neuronal injury cycle. Activated mitochondrial apoptotic pathway, characterized by increased caspase-3 and caspase-9 cleavage and Bax/Bcl2 ratio in the hippocampal and cortical neurons, stimulate neurocognitive impairments. Additionally, altered LC3-II/I and P62/SQSTM1, p-mTOR, p-AMPK1 and p-ULK1 levels and dysregulated autophagosome-lysosome fusion decrease neuronal and glial energy homeostasis. The therapies and procedures for attenuating sepsis-induced brain damage include early resuscitation, cerebral blood flow autoregulation, implantable electric vagus nerve stimulation, antioxidants, statins, glucocorticoids, neuroimmune axis modulators and PKCδ inhibitors. The current review enumerates the pathophysiology of sepsis-induced brain damage, its diagnosis, the role of critical inducers and mediators and, ultimately, therapeutic measures attenuating cerebrovascular degeneration.

Obligatory role for PKCδ in PIP2 -mediated activation of store-operated TRPC1 channels in vascular smooth muscle cells.

Key points In vascular smooth muscle cells (VSMCs), activation of Ca2+‐permeable store‐operated channels (SOCs) composed of canonical transient receptor potential channel 1 (TRPC1) subunits mediates Ca2+ entry pathways that regulate contraction, proliferation and migration, which are processes associated with vascular disease.Activation of TRPC1‐based SOCs requires protein kinase C (PKC) activity, which is proposed to phosphorylate TRPC1 proteins to promote channel opening by phosphatidylinositol 4,5‐bisphosphate (PIP2). We investigated the identity of the PKC isoform involved in activating TRPC1‐based SOCs in rat mesenteric artery VSMCs.TRPC1‐based SOCs were reduced by PKCδ inhibitors and knockdown of PKCδ expression. Store depletion induced interactions between TRPC1 and PKCδ and PKCδ‐dependent phosphorylation of TRPC1. Furthermore, generation of store‐operated interactions between PIP2 and TRPC1 and activation of TRPC1‐based SOCs by PIP2 required PKCδ.These findings reveal that PKCδ activity has an obligatory role in activating TRPC1‐based SOCs, through regulating PIP2‐mediated channel opening. In vascular smooth muscle cells (VMSCs), stimulation of Ca2+‐permeable canonical transient receptor potential channel 1 (TRPC1)‐based store‐operated channels (SOCs) mediates Ca2+ entry pathways that regulate cell contraction, proliferation and migration, which are processes associated with vascular disease. It is therefore important to understand how TRPC1‐based SOCs are activated. Stimulation of TRPC1‐based SOCs requires protein kinase C (PKC) activity, with store‐operated PKC‐dependent phosphorylation of TRPC1 essential for channel opening by phosphatidylinositol 4,5‐bisphosphate (PIP2). Experimental protocols used to activate TRPC1‐based SOCs suggest that the PKC isoform involved requires diacylglycerol (DAG) but is Ca2+‐insensitive, which are characteristics of the novel group of PKC isoforms (δ, ε, η, θ). Hence, the present study examined whether a novel PKC isoform(s) is involved in activating TRPC1‐based SOCs in contractile rat mesenteric artery VSMCs. Store‐operated whole‐cell cation currents were blocked by Pico145, a highly selective and potent TRPC1/4/5 channel blocker and T1E3, a TRPC1 blocking antibody. PKCδ was expressed in VSMCs, and selective PKCδ inhibitory peptides and knockdown of PKCδ expression with morpholinos oligomers inhibited TRPC1‐based SOCs. TRPC1 and PKCδ interactions and phosphorylation of TRPC1 induced by store depletion were both reduced by pharmacological inhibition and PKCδ knockdown. In addition, store‐operated PIP2 and TRPC1 interactions were blocked by PKCδ inhibition, and PKCδ was required for PIP2‐mediated activation of TRPC1 currents. These results identify the involvement of PKCδ in stimulation of TRPC1‐based SOCs and highlight that store‐operated PKCδ activity is obligatory for channel opening by PIP2, the probable activating ligand.

TRPV4 promotes acoustic wave-mediated BBB opening via Ca2+/PKC-δ pathway

IntroductionNumerous studies have shown the ability of low-energy acoustic waves such as focused ultrasound or shockwave to transiently open blood-brain barrier (BBB) and facilitate drug delivery to the brain. Preclinical and clinical evidences have well demonstrated the efficacy and safety in treating various brain disorders. However, the molecular mechanisms of acoustic waves on the BBB are still not fully understood. ObjectivesThe present study aimed at exploring the possible molecular mechanisms of acoustic wave stimulation on brains. Methods: Briefly describe the experimental designThe left hemisphere of the rat‘s brain was treated with pulsed ultrasound from a commercial focused shockwave or a planar ultrasound device, and the right hemisphere served as a control. One hour after the mechanical wave stimulation or overnight, the rats were sacrificed and the brains were harvested for protein or histological analysis. Agonists and antagonists related to the signal transduction pathways of tight junction proteins were used to investigate the possible intracellular mechanisms. ResultsIntracellular signal transduction analysis shows calcium influx through transient receptor potential vanilloid 4 (TRPV4) channels, and the activation of PKC-δ pathway to mediate dissociation of ZO-1 and occludin after acoustic wave stimulation. The activation of TRPV4 or PKC-δ signaling further increased the expression level of TRPV4, suggesting a feedback loop to regulate BBB permeability. Moreover, the tight junction proteins dissociation can be reversed by administration of PKC-δ inhibitor and TRPV4 antagonist. ConclusionThe present study shows the crucial role of TRPV4 in acoustic wave-mediated BBB permeability, specifically its effect on compromising tight junction proteins, ZO-1 and occludin. Our findings provide a new molecular perspective to explain acoustic wave-mediated BBB opening. Moreover, activation of TRPV4 by agonists may reduce the threshold intensity level of acoustic waves for BBB opening, which may prevent undesirable mechanical damages while maintaining efficient BBB opening.

Wenxin Granule Ameliorates Hypoxia/Reoxygenation-Induced Oxidative Stress in Mitochondria via the PKC-δ/NOX2/ROS Pathway in H9c2 Cells.

Myocardial infarction and following reperfusion therapy-induced myocardial ischemia/reperfusion (I/R) injury have been recognized as an important subject of cardiovascular disease with high mortality. As the antiarrhythmic agent, Wenxin Granule (WXG) is widely used to arrhythmia and heart failure. In our pilot study, we found the antioxidative potential of WXG in the treatment of myocardial I/R. This study is aimed at investigating whether WXG could treat cardiomyocyte hypoxia/reoxygenation (H/R) injury by inhibiting oxidative stress in mitochondria. The H9c2 cardiomyocyte cell line was subject to H/R stimuli to mimic I/R injury in vitro. WXG was added to the culture medium 24 h before H/R exposing as pretreatment. Protein kinase C-δ (PKC-δ) inhibitor rottlerin or PKC-δ lentivirus vectors were conducted on H9c2 cells to downregulate or overexpress PKC-δ protein. Then, the cell viability, oxidative stress levels, intracellular and mitochondrial ROS levels, mitochondrial function, and apoptosis index were analyzed. In addition, PKC-δ protein expression in each group was verified by western blot analysis. Compared with the control group, the PKC-δ protein level was significantly increased in the H/R group, which was remarkably improved by WXG or rottlerin. PKC-δ lentivirus vector-mediated PKC-δ overexpression was not reduced by WXG. WXG significantly improved H/R-induced cell injury, lower levels of SOD and GSH/GSSG ratio, higher levels of MDA, intracellular and mitochondrial ROS content, mitochondrial membrane potential and ATP loss, mitochondrial permeability transition pore opening, NOX2 activation, cytochrome C release, Bax/Bcl-2 ratio and cleaved caspase-3 increasing, and cell apoptosis. Similar findings were obtained from rottlerin treatment. However, the protective effects of WXG were abolished by PKC-δ overexpression, indicating that PKC-δ was a potential target of WXG treatment. Our findings demonstrated a novel mechanism by which WXG attenuated oxidative stress and mitochondrial dysfunction of H9c2 cells induced by H/R stimulation via inhibitory regulation of PKC-δ/NOX2/ROS signaling.

Searching for combination therapy by clustering methods: Stimulation of PKC in Golgi apparatus combined with hypericin induced PDT.

Cancer cell metabolism is a very attractive target for anticancer treatments. This work focuses on protein kinase C (PKC) signaling in the U87 MG glioma. By means of western blot, fluorescence and time-resolved fluorescence microscopy the correlation between the Golgi apparatus (GA), lysosomes and mitochondria were evaluated. The known regulators of PKC were applied to cancer cells. Phorbol myristate acetate (PMA) was chosen as the activator of PKC. Gö6976, hypericin and rottlerin, the inhibitors of PKCα and PKCδ were selected as well. Stabilization, destabilization processes occurring in cells allow classification of observations into several groups. Multiple versions of hierarchical cluster analysis have been applied and similarities have been found between organelles and PKC regulators. The method identified GA as an extraordinary organelle whose functionality is significantly influenced by PKC regulators as well as oxidative stress. Therefore, combination therapy has been designed according to the results of the cluster analysis. Furthermore, the efficacy of photodynamic therapy mediated by hypericin, and the consequent apoptosis, was significantly increased during the treatment. To our knowledge, this is the first demonstration of the effectiveness of the clustering in the given area.

Suppression of PKCδ/NF-κB Signaling and Apoptosis Induction through Extrinsic/Intrinsic Pathways Are Associated Magnolol-Inhibited Tumor Progression in Colorectal Cancer In Vitro and In Vivo.

Magnolol is one of the hydroxylated biphenyl compounds from the root and stem bark of Magnolia officinalis, which shown to possess anti-colorectal cancer (CRC) effects. However, the regulatory mechanism of magnolol on apoptosis and NF-κB signaling in human CRC has not been elucidated. Thus, we investigated the inhibitory mechanism of magnolol on human and mouse CRC (HT-29 and CT-26) in vitro and in vivo. Results from reporter gene assay indicated that both magnolol and rottlerin (PKCδ inhibitor) reduced the endogenous NF-κB activity. In addition, indolactam V (PKCδ activator)-induced NF-κB signaling was significantly suppressed with both magnolol and rottlerin treatment. Results from Western blotting also indicated that phosphorylation of PKCδ and NF-κB -related proteins involved in tumor progression were effectively decreased by magnolol treatment. The invasion capacity of CRC cells was also attenuated by both magnolol and rottlerin. Furthermore, magnolol triggered Fas/Fas-L mediated extrinsic apoptosis and mitochondria mediated intrinsic apoptosis were validated by flow cytometry. Most importantly, tumor growth in both HT-29 and CT-26 bearing mice were suppressed by magnolol, but no pathologic change was detected in mice kidney, spleen, and liver. As confirmed by immunohistochemistry (IHC) staining from tumor tissue, PKCδ/NF-κB signaling and downstream proteins expression were decreased, while apoptotic proteins expression was increased in the magnolol treated group. According to these results, we suggest that the induction of apoptosis through extrinsic/intrinsic pathways and the blockage of PKCδ/NF-κB signaling are associated with the magnolol-inhibited progression of CRC.

5-HT2A receptor-mediated PKCδ phosphorylation is critical for serotonergic impairments induced by p-chloroamphetamine in mice

p-Chloroamphetamine (PCA), an amphetamine derivative, has been shown to induce serotonergic toxicity. However, the precise mechanism of serotonergic toxicity induced by PCA remains unclear. In this study, PCA treatment (20 mg/kg, i.p.) did not significantly change 5-HT1A receptor gene expression, but significantly increased 5-HT2A receptor gene expression. Furthermore, 5-HT2A receptor antagonist MDL11939, but not 5-HT1A receptor antagonist WAY100635, significantly attenuated PCA-induced serotonergic impairments. We investigated whether PCA activated a specific isoform of protein kinase C (PKC), since previous evidence indicated the involvement of PKC in neurotoxicity induced by amphetamines. We observed that PCA treatment significantly increased the expression levels of PKCδ among all PKC isoforms. MDL11939 treatment significantly attenuated PCA-induced phosphorylation of PKCδ. However, PCA-induced increase in 5-HT2A receptor gene expression was not altered by rottlerin (a pharmacological inhibitor of PKCδ) in mice, suggesting that 5-HT2A receptor is an upstream molecule for the activation of PKCδ. Rottlerin or PKCδ knockout significantly attenuated serotonergic behaviors. However, MDL11939 did not show any additional effects against the attenuation caused by PKCδ knockout in mice, suggesting that PKCδ gene is a molecular target for 5-HT2A receptor-mediated serotonergic effects. Our results suggest that 5-HT2A receptor mediates PCA-induced serotonergic impairments via activation of PKC.δ.

Protein Kinase C-δ Mediates Kidney Tubular Injury in Cold Storage-Associated Kidney Transplantation.

Kidney injury associated with cold storage is a determinant of delayed graft function and the long-term outcome of transplanted kidneys, but the underlying mechanism remains elusive. We previously reported a role of protein kinase C-δ (PKCδ) in renal tubular injury during cisplatin nephrotoxicity and albumin-associated kidney injury, but whether PKCδ is involved in ischemic or transplantation-associated kidney injury is unknown. To investigate PKCδ's potential role in injury during cold storage-associated transplantation, we incubated rat kidney proximal tubule cells in University of Wisconsin (UW) solution at 4°C for cold storage, returning them to normal culture medium at 37°C for rewarming. We also stored kidneys from donor mice in cold UW solution for various durations, followed by transplantation into syngeneic recipient mice. We observed PKCδ activation in both in vitro and in vivo models of cold-storage rewarming or transplantation. In the mouse model, PKCδ was activated and accumulated in mitochondria, where it mediated phosphorylation of a mitochondrial fission protein, dynamin-related protein 1 (Drp1), at serine 616. Drp1 activation resulted in mitochondrial fission or fragmentation, accompanied by mitochondrial damage and tubular cell death. Deficiency of PKCδ in donor kidney ameliorated Drp1 phosphorylation, mitochondrial damage, tubular cell death, and kidney injury during cold storage-associated transplantation. PKCδ deficiency also improved the repair and function of the renal graft as a life-supporting kidney. An inhibitor of PKCδ, δV1-1, protected kidneys against cold storage-associated transplantation injury. These results indicate that PKCδ is a key mediator of mitochondrial damage and renal tubular injury in cold storage-associated transplantation and may be an effective therapeutic target for improving renal transplant outcomes.

Diallyl Trisulfide (DATS) Suppresses AGE-Induced Cardiomyocyte Apoptosis by Targeting ROS-Mediated PKCδ Activation.

Chronic high-glucose exposure results in the production of advanced glycation end-products (AGEs) leading to reactive oxygen species (ROS) generation, which contributes to the development of diabetic cardiomyopathy. PKCδ activation leading to ROS production and mitochondrial dysfunction involved in AGE-induced cardiomyocyte apoptosis was reported in our previous study. Diallyl trisulfide (DATS) is a natural cytoprotective compound under various stress conditions. In this study, the cardioprotective effect of DATS against rat streptozotocin (STZ)-induced diabetic mellitus (DM) and AGE-induced H9c2 cardiomyoblast cell/neonatal rat ventricular myocyte (NRVM) damage was assessed. We observed that DATS treatment led to a dose-dependent increase in cell viability and decreased levels of ROS, inhibition of PKCδ activation, and recuded apoptosis-related proteins. Most importantly, DATS reduced PKCδ mitochondrial translocation induced by AGE. However, apoptosis was not inhibited by DATS in cells transfected with PKCδ-wild type (WT). Inhibition of PKCδ by PKCδ-kinase-deficient (KD) or rottlerin not only inhibited cardiac PKCδ activation but also attenuated cardiac cell apoptosis. Interestingly, overexpression of PKCδ-WT plasmids reversed the inhibitory effects of DATS on PKCδ activation and apoptosis in cardiac cells exposed to AGE, indicating that DATS may inhibit AGE-induced apoptosis by downregulating PKCδ activation. Similar results were observed in AGE-induced NRVM cells and STZ-treated DM rats following DATS administration. Taken together, our results suggested that DATS reduced AGE-induced cardiomyocyte apoptosis by eliminating ROS and downstream PKCδ signaling, suggesting that DATS has potential in diabetic cardiomyopathy (DCM) treatment.

The Combination of Astragalus membranaceus and Ligustrazine Protects Against Thrombolysis-Induced Hemorrhagic Transformation Through PKCδ/Marcks Pathway in Cerebral Ischemia Rats

Astragalus membranaceus (Ast) and ligustrazine (Lig) have a protective effect on lower hemorrhagic transformation induced by pharmaceutical thrombolysis. The cerebral ischemia rat model was induced with autologous blood clot injections. A combination of Ast and Lig, or a protein kinase C delta (PKCδ) inhibitor—rottlerin, or a combination of Ast, Lig, and rottlerin was administered immediately after recombinant tissue plasminogen activator injection. The cerebral infarct area, neurological deficits, cerebral hemorrhage status, neuronal damage and tight junctions’ changes in cerebral vessels, and the messenger RNA and protein levels of PKCδ, myristoylated alanine-rich C kinase substrate (Marcks), and matrix metallopeptidase 9 (MMP9) were determined after 3 h and 24 h of thrombolysis. The ultrastructure of the neuronal damage and tight junctions was examined under a transmission electron microscope. The expression levels of PKCδ, Marcks, and MMP9 were assessed by immunohistochemistry, western blot, and quantitative real-time polymerase chain reaction . Administration of Ast and Lig not only significantly decreased neurological deficit scores, infarct volumes, and cerebral hemorrhage but also inhibited the disruption due to neuronal dysfunction and the tight junction integrity in the cerebral vessel. Treatment with a combination of Ast and Lig effectively protected ischemia-induced microhemorrhage transformation through PKCδ/Marcks pathway suppression.

Protein kinase C-delta inhibition is organ-protective, enhances pathogen clearance, and improves survival in sepsis.

Sepsis is a leading cause of morbidity and mortality in intensive care units. Previously, we identified Protein Kinase C-delta (PKCδ) as an important regulator of the inflammatory response in sepsis. An important issue in development of anti-inflammatory therapeutics is the risk of immunosuppression and inability to effectively clear pathogens. In this study, we investigated whether PKCδ inhibition prevented organ dysfunction and improved survival without compromising pathogen clearance. Sprague Dawley rats underwent sham surgery or cecal ligation and puncture (CLP) to induce sepsis. Post-surgery, PBS or a PKCδ inhibitor (200µg/kg) was administered intra-tracheally (IT). At 24hours post-CLP, there was evidence of lung and kidney dysfunction. PKCδ inhibition decreased leukocyte influx in these organs, decreased endothelial permeability, improved gas exchange, and reduced blood urea nitrogen/creatinine ratios indicating organ protection. PKCδ inhibition significantly decreased bacterial levels in the peritoneal cavity, spleen and blood but did not exhibit direct bactericidal properties. Peritoneal chemokine levels, neutrophil numbers, or macrophage phenotypes were not altered by PKCδ inhibition. Peritoneal macrophages isolated from PKCδ inhibitor-treated septic rats demonstrated increased bacterial phagocytosis. Importantly, PKCδ inhibition increased survival. Thus, PKCδ inhibition improved survival and improved survival was associated with increased phagocytic activity, enhanced pathogen clearance, and decreased organ injury.

Quercetin improves endothelial insulin sensitivity in obese mice by inhibiting Drp1 phosphorylation at serine 616 and mitochondrial fragmentation.

Studies have shown that endothelial insulin resistance induced by oxidative stress contributes to vascular dysfunction in metabolic disorders. Quercetin, a natural antioxidant, has been recently shown to exert protective effects on endothelial function. However, the effects of quercetin on endothelial insulin resistance and its underlying mechanism are unclear. Here, we found that chronic oral treatment of obese mice with quercetin increased vascular endothelial insulin sensitivity, accompanied by alleviated mitochondrial fragmentation as revealed by confocal imaging. In addition, western blot analysis showed that quercetin treatment suppressed the levels of dynamin-related protein 1 (Drp1) and phosphorylation at serine 616 in endothelial cells of obese mice. Mechanistically, quercetin specifically suppressed Drp1 phosphorylation at serine 616, whereas it showed little effects on the Drp1 level and its phosphorylation at serine 637 in cultured endothelial cells under oxidative stress. Furthermore, our results also showed that quercetin suppressed Drp1 phosphorylation at serine 616 by inhibiting PKCδ as revealed by western blot analysis. Knockdown of PKCδ with siRNA alleviated the protective effects of quercetin on endothelial-mitochondrial dynamics and insulin sensitivity. These results suggest that chronic oral treatment with quercetin exerts endothelial protective effects through inhibition of PKCδ and the resultant mitochondrial fragmentation.

PKCδ Mediates NF-κB Inflammatory Response and Downregulates SIRT1 Expression in Liver Fibrosis.

The precise mechanism of hepatic cirrhosis remains largely unclear. In particular, a potential regulatory mechanism by which protein kinase C-delta (PKCδ ) affects profibrogenic gene expression involved in hepatic cirrhosis has never been explored. In the present study, we investigated whether PKCδ activation is involved in liver inflammatory fibrosis in both lipopolysaccharide (LPS)-treated RAW 264.7 and CCl4-treated mice. PKCδ was strongly activated by LPS or CCl4 treatment and consequently stimulated nuclear factor (NF)-κB inflammatory response. Interestingly, the activation of PKCδ negatively regulated sirtuin-1 (SIRT1) expression, whereas PKCδ suppression by PKCδ peptide inhibitor V1-1 or siRNA dramatically increased SIRT1 expression. Furthermore, we showed that the negative regulation of PKCδ leads to a decrease in SIRT1 expression. To our knowledge, these results are the first demonstration of the involvement of PKCδ in modulating NF-κB through SIRT1 signaling in fibrosis in mice, suggesting a novel role of PKCδ in inflammatory fibrosis. The level of NF-κB p65 in the nucleus was also negatively regulated by SIRT1 activity. We showed that the inhibition of PKCδ promoted SIRT1 expression and decreased p65 levels in the nucleus through deacetylation. Moreover, the inactivation of PKCδ with V1-1 dramatically suppressed the inflammatory fibrosis, indicating that PKCδ represents a promising target for treating fibrotic diseases like hepatic cirrhosis.

PKCδ promotes fertilization of mouse embryos in early development via the Cdc25B signaling pathway.

Protein kinase C type δ (PKCδ) is involved in B-cell signaling and the regulation of growth, apoptosis and differentiation of a variety of cell types. Cell division cycle 25 (Cdc25) is a key mediator of cell cycle progression that activates cyclin-dependent kinase complexes that drive the cell cycle and participates in the regulation of DNA damage checkpoints. Cdc25B is a member of the Cdc25 family of phosphatases. The present study investigated the role and mechanism of PKCδ in regulating the fertilization of mouse embryos in early development. The expression and subcellular localization of PKCδ and Cdc25B were detected using reverse transcription-quantitative polymerase chain reaction, western blotting and immunofluorescence in one-cell stage mouse embryos. Specific small interfering RNAs targeting PKCδ were used to knockdown the expression of PKCδ. Subsequently, Scansite software was used to predict the target of phosphorylated Cdc25B. Western blotting was used to measure the effects of phosphorylation and dephosphorylation in one-cell stage mouse embryos at different cell cycle phases. PKCδ was expressed during M phase and served a positive role in one-cell stage mouse embryos. Immunofluorescence data revealed that PKCδ and Cdc25B were expressed during G1, S, G2 and M phases of the cell cycle. Furthermore, phosphorylated levels of Cdc25B-Ser96 were observed during G2 and M phases. Microinjection with mimics of phosphorylated Cdc25B-Ser96 mRNA promoted the development of one-cell stage mouse embryos. When PKCδ was suppressed, microinjection with mimics of phosphorylated Cdc25B-Ser96 mRNA reversed the inhibition of PKCδ. To conclude, PKCδ serves a positive role in the first cell cycle of mouse embryos by phosphorylating Cdc25B-Ser96, and provides novel insights for the regulation of early embryonic development.

Toxoplasma gondii activates a Syk-CARD9-NF-κB signaling axis and gasdermin D-independent release of IL-1β during infection of primary human monocytes.

IL-1β is a potent pro-inflammatory cytokine that promotes immunity and host defense, and its dysregulation is associated with immune pathology. Toxoplasma gondii infection of myeloid cells triggers the production and release of IL-1β; however, the mechanisms regulating this pathway, particularly in human immune cells, are incompletely understood. We have identified a novel pathway of T. gondii induction of IL-1β via a Syk-CARD9-NF-κB signaling axis in primary human peripheral blood monocytes. Syk was rapidly phosphorylated during T. gondii infection of primary monocytes, and inhibiting Syk with the pharmacological inhibitors R406 or entospletinib, or genetic ablation of Syk in THP-1 cells, reduced IL-1β release. Inhibition of Syk in primary cells or deletion of Syk in THP-1 cells decreased parasite-induced IL-1β transcripts and the production of pro-IL-1β. Furthermore, inhibition of PKCδ, CARD9/MALT-1 and IKK reduced p65 phosphorylation and pro-IL-1β production in T. gondii-infected primary monocytes, and genetic knockout of PKCδ or CARD9 in THP-1 cells also reduced pro-IL-1β protein levels and IL-1β release during T. gondii infection, indicating that Syk functions upstream of this NF-κB-dependent signaling pathway for IL-1β transcriptional activation. IL-1β release from T. gondii-infected primary human monocytes required the NLRP3-caspase-1 inflammasome, but interestingly, was independent of gasdermin D (GSDMD) cleavage and pyroptosis. Moreover, GSDMD knockout THP-1 cells released comparable amounts of IL-1β to wild-type THP-1 cells after T. gondii infection. Taken together, our data indicate that T. gondii induces a Syk-CARD9/MALT-1-NF-κB signaling pathway and activation of the NLRP3 inflammasome for the release of IL-1β in a cell death- and GSDMD-independent manner. This research expands our understanding of the molecular basis for human innate immune regulation of inflammation and host defense during parasite infection.

Protein kinase Cδ mediates methamphetamine-induced dopaminergic neurotoxicity in mice via activation of microsomal epoxide hydrolase

We previously demonstrated that activation of protein kinase Cδ (PKCδ) is critical for methamphetamine (MA)-induced dopaminergic toxicity. It was recognized that microsomal epoxide hydrolase (mEH) also induces dopaminergic neurotoxicity. It was demonstrated that inhibition of PKC modulates the expression of mEH. We investigated whether MA-induced PKCδ activation requires mEH induction in mice. MA treatment (8 mg/kg, i.p., × 4; 2 h interval) significantly enhanced the level of phosphorylated PKCδ in the striatum of wild type (WT) mice. Subsequently, treatment with MA resulted in significant increases in the expression of cleaved PKCδ and mEH. Treatment with MA resulted in enhanced interaction between PKCδ and mEH. PKCδ knockout mice exhibited significant attenuation of the enhanced mEH expression induced by MA. MA-induced hyperthermia, oxidative stress, proapoptotic potentials, and dopaminergic impairments were attenuated by PKCδ knockout or mEH knockout in mice. However, treating mEH knockout in mice with PKCδ inhibitor, rottlerin did not show any additive beneficial effects, indicating that mEH is a critical mediator of neurotoxic potential of PKCδ. Our results suggest that MA-induced PKCδ activation requires mEH induction as a downstream signaling pathway and that the modulation of the PKCδ and mEH interaction is important for the pharmacological intervention against MA-induced dopaminergic neurotoxicity.